Heterogeneous immunoassays typically are performed using a solid support, preferably with the solid support being formed from magnetic particles. A particularly preferred support for this purpose is that described in U.S. Pat. No. 4,661,408 assigned to E.I. du Pont de Nemours & Co., Inc. This patent describes a chromium dioxide particle which has favorable magnetic properties for use as a solid support in such assays.
The concept of using magnetically responsive particles to effect separations of bioactive materials is old in the art (Hedin, C.G., Biotech. Bioeng. Symp. No. 3 (1972) 173-174; Robinson, P.J., et al., Biotech Bioeng. (1973) 15, 603-606). The concept has been extended over time to include affinity purification of enzymes, proteins or microorganisms applicable to any sorption-desorption process (Dunhill, P., et al., Biotech. Bioeng. (1974) 10, 987-990; Horisberger, M., Biotech. Bioeng. (1976) 18, 1647-1651).
Another improved magnetically responsive particle is described by Mansfield et al. in U.S. Pat. No. 4,197,337. These particles are porous glass microparticles with magnetic material imbedded within them. This gives the particles the properties of high surface area, inertness and being substantially superparamagnetic. This high surface area again favors rapid reaction kinetics and increases capacity of the individual particles. Being substantially superparamagnetic, basically means the particles do not retain much magnetic memory, or retentivity, when removed from a magnetic field. This means that particles can be repeatedly separated from their environment by a magnetic field without affecting the ability to redisperse those particles. This is of advantage in sandwich immunoassays where multiple washing steps may require repeated separation and redispersion.
The protected CrO.sub.2 particles described in the Du Pont patent have several properties that are particularly advantageous in heterogeneous immunoassays. These are:
low remanent magnetism and favorable surface structure--allowing repeated magnetic separation/dispersion cycles; rapid separation in a magnetic field; high surface area for high capture capacity; a highly stable particle for maximum reagent shelf life.
One problem with immunoassays is that they require repeated washing of the solid support containing the bound component following removal of the free component. This is a particularly difficult procedure even when performed manually. It is a particular problem when the automatic wash procedure is incorporated into an automatic instrument which is capable of performing immunoassays. The typical requirements for purity in such immunoassays is that the bound component remaining following such washes should not contain more than 20 parts per million of the original sample/conjugate matrix. This necessitates the use of multiple wash stations and can be accomplished in automatic instruments by providing such multiple wash stations. However, the several mechanisms required to operate individually the several wash stations and the mechanisms required for the wash stations themselves can become quite expensive.
There are essentially two types of heterogeneous immunoassays. These are competitive immunoassays and sandwich immunoassays. In a competitive assay, an antibody to an antigen contained in a first reagent is attached to the derivatized magnetic particles to make up a solid phase. The second reagent, consisting of antigen attached to a tag (a measurable entity, including radioactive molecules, fluorescent molecules, or enzymes), and patient sample are mixed with the solid phase in a test tube. In the absence of patient antigen, some 50% of the antigen-tag is bound to the antibody of the magnetic solid phase. In the presence of patient antigen, some of the antibodies are filled up with patient antigen and are unavailable to the tag antigen. As a result, increasing amount of patient antigen leads to decreasing amount of tag antigen. Thus one can form a calibration chart relating the amount of patient antigen to the amount of tag antigen. The separation and wash stages results from the need to measure the free tag or the bound tag, not the total tag added. The magnetic particle facilitates this separation by forming the particles with the bound tag into a pellet on the side of the tube. The free tag can then be removed as by aspiration. Following the separation and removal of free tag, another reagent is added so that the amount of bound tag can be measured. In a typical case, enzyme is used as the tag so that the reagent added is a substrate for the enzyme permitting the measurement of the amount of tag that was bound to antibody.
In a typical sandwich immunoassay, an antibody to an antigen is attached to the magnetic particle. This is in high concentration relative to the amount of patient antigen in a sample. Patient antigen is captured by the antibody on the magnetic particles and then the particles (and captured patient antigen) separated from interfering substances in the sample. To this, a second reagent, containing a second antibody with an attached tag, is added. This second antibody attaches to the patient antigen, captured by the first antibody on the magnetic particle, and results in the formation of a sandwich so that the second antibody tag is held firmly by the antigen to the first antibody on the magnetic particle. At this point, a magnetic separation similar to that described, permits the determination of bound tag which is in proportion to the patient antigen, the excess tag of the second reagent having been removed by aspiration.
Magnetic particles are particularly useful as the solid support in heterogeneous immunoassay because they can readily separate the free from the bound tag. Such immunoassays using magnetic particles as a solid support are described for example in U.S. Pat. No. 4,661,408 (Lau et al.), U.S. Pat. No. 4,628,037 issued to Chagnon et al., U.S. Pat. No. 4,672,040 issued to Josephson, and U.S. Pat. No. 4,698,302 issued to Whitehead et al. The methods disclosed in all of these patents separation units such as those that are available from Corning Medical, Corning Glass Works, Medfield, Mass. Such manual techniques are relatively slow, require relatively strong magnets which are expensive, require considerable manual dexterity, and require an excessive amount of time to effect the separation with the purity required, particularly for sandwich type heterogeneous immunoassays.
There are numerous automatic clinical analysis type instruments available on the market today. Typical of these is the Automatic Clinical Analyzer known as the aca.RTM. sold by E.I. du Pont de Nemours & Co., Inc. of Wilmington, Del. This is an instrument in which an incubator is used to process samples. The incubator is in the form of a belt or chain in which a sample mixed with reagents in packs and is analyzed for its various components. While quite satisfactory for many purposes it does not have the versatility required for some of the more recently developed sensitive immunoassays.
Other analyzers that are available for analyzing samples are those that are described, for example, in U.S. Pat. No. 4,315,891 assigned to Olympus Optical Company. This patent describes a belt or chain incubator and has a single reaction line in which reaction vessels are carried step by step along the reaction line. Samples and reagents are delivered to the vessels during their movement along the reaction line to obtain a test liquid which is then subjected to photometric analysis. This machine permits several tests to be run simultaneously on samples by an interleaving processing. Unfortunately, this apparatus does not have the capability for performing the precise washing required in heterogeneous immunoassays. The apparatus also requires a relatively large amount of space.
Another automatic analytical apparatus is that described in U.S. Pat. No. 4,459,265 assigned to Clinicon. This apparatus includes a stepwise rotatable circular plate. It carries a plurality of reaction tubes on its periphery with several reagent supply stations arranged at different locations around such periphery. The use of multiple stations does provide the machine with the versatility to carry out several different test methodologies, but again does not provide the necessary washing required for the more sensitive heterogeneous immunoassays.
Many of the machines described use circular trays that rotate to provide the necessary random access to add sample and reagent to, incubate, wash the magnetic particles, etc., in the reaction vessels. This creates the need for greater space requiring larger instruments that would otherwise be desirable. It is also necessary in such instruments to have random access of the samples to be processed to the real time of the immunoassay system.